PROJECT SUMMARY/ABSTRACT Understanding the neural systems specifically driving compulsive drug seeking is essential for developing treatments for addiction as a disease. The orexin/hypocretin (ORX) system in the hypothalamus has been implicated in motivation for natural and drug rewards, in addition to its role in arousal and other physiological functions. This system may play a critical role in the extremely high levels of motivation and craving underlying addiction, but it is unknown how specific populations of ORX neurons contribute to compulsive drug seeking. Different ORX neuron populations may regulate different behavioral or physiological functions based on anatomical location or connectivity. Identification of specific neuron ensembles that selectively regulate compulsive drug seeking will allow the development of treatments for drug craving that do not affect other ORX-mediated functions such as arousal or normal hedonic states. Here we will investigate the roles of individual ORX neurons in rat models of compulsive drug and natural reward (sucrose) seeking. We will selectively express optogenetic and chemogenetic effectors in ORX neurons to monitor and modulate specific ORX neuron populations during cocaine and sucrose seeking. In Aim 1 we will use multi-neuron ensemble recording combined with optogenetic tagging to characterize the activity of ORX neurons during cocaine and sucrose seeking. In Aim 2 we will selectively modulate either lateral or medial hypothalamic subpopulations of ORX neurons during cocaine and sucrose seeking using designer receptors exclusively activated by designer drugs (DREADDs). The studies will focus on regulated and compulsive models of drug and sucrose seeking to examine changes in ORX function as compulsive drug/reward seeking develops. Based on previous studies and preliminary results we hypothesize that lateral ORX neurons will encode and regulate drug/reward seeking early in regulated seeking, but that medial regions will also be recruited in late-stage compulsive seeking, particularly of cocaine. These studies are the first in a series designed to precisely identify unique functions of specific ORX neuron populations based on a wide range of factors, and to determine their unique contributions to addiction and related compulsive behaviors.